Daniel Aaen Hansen

Plasmodium falciparum Erythrocyte Membrane Protein 1 Diversity in Seven Genomes – Divide and Conquer

The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A–E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a platform for future studies on var/PfEMP1 expression and function.

Proteomics Analysis of Cytokine-induced Dysfunction and Death in Insulin-producing INS-1E Cells New Insights into the Pathways Involved

Cytokines released by islet-infiltrating immune cells play a crucial role in β-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this β-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 ± 164 (pH 4–7) and 1641 ± 73 (pH 6–9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p ≤ 0.01) by a combination of interleukin-1β and interferon-γ, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e−05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a β-cell dysfunction/apoptotic β-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to β-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of β-cell loss in type 1 diabetes.

Differential Protein Pathways in 1,25-Dihydroxyvitamin D-3 and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)(2)D(3)), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)(2)D(3), dexamethasone (DEX), and a combination of both compounds (combi). Human CD14(+) monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)(2)D(3) and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3-7NL and 6-9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)(2)D(3)- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)(2)D(3) is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)(2)D(3) with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)(2)D(3) effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)(2)D(3), rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)(2)D(3) and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.

Proteome analysis demonstrates profound alterations in human dendritic cell nature by TX527, an analogue of vitamin D.

Structural analogues of vitamin D have been put forward as therapeutic agents able to exploit the immunomodulatory effects of vitamin D, without its undesired calcemic side effects. We have demonstrated that TX527 affects dendritic cell (DC) maturation in vitro, resulting in the generation of a tolerogenic cell. In the present study, we aimed to explore the global protein changes induced by the analogue in immature DC (iDC) and mature human DC and to correlate them with alterations in DC morphology and function. Human CD14+ monocytes were differentiated toward iDC or mature DCs, in the presence or absence of TX527 (10−8 M) (n=4). Protein samples were separated into two different pH ranges (pH4–7 and 6–9), analyzed by 2‐D DIGE and differentially expressed spots (p<0.01) were identified by MALDI‐TOF/TOF (76.3 and 70.7% in iDC and mature DCs, respectively). Differential protein expression revealed three protein groups predominantly affected by TX527 treatment, namely proteins involved in cytoskeleton structure, in protein biosynthesis/proteolysis and in metabolism. Moreover, protein interactome‐network analysis demonstrated close interaction between these different groups (p<0.001) and morphological and functional analyses confirmed the integrated effect of TX527 on human DCs, resulting in a cell with altered morphology, cell surface marker expression, endocytic and migratory capacity.

Novel Insights into the Global Proteome Responses of Insulin-Producing INS-1E Cells To Different Degrees of Endoplasmic Reticulum Stress

Exposure of insulin-secreting β-cells to inflammatory cytokines or high concentrations of free fatty acids, factors involved in the pathogenesis of type 1 and type 2 diabetes, leads to endoplasmic reticulum (ER) stress, β-cell dysfunction, and eventually apoptotic β-cell death. The aim of this study was to investigate the impact of ER stress on β-cells at the protein level to evaluate the contribution of post-transcriptional and post-translational changes in ER stress-induced β-cell damage. INS-1E cells were exposed in vitro to the ER-stress inducer cyclopiazonic acid (CPA) at two concentrations, and protein changes were evaluated using 2D-DIGE. CPA, 25 μM, led to massive apoptosis, accompanied by a near complete protein translation shut-down. CPA, 6.25 μM, led to adaptation of the β-cells to ER stress. Identification of the differentially expressed proteins in the two conditions led to the discovery of a clear pattern of defense pathways, with post-translational modifications playing a crucial role. Key alterations included inhibition of insulin translation and post-translational modifications in ER chaperones HYOU1 and HSPA5. Also, a central role for 14-3-3 proteins is suggested. In conclusion, INS-1E cells are highly sensitive to ER stress, leading to important post-transcriptional and post-translational modifications that may contribute to β-cell dysfunction and death.

Uncovering the Molecular Machinery of the Human Spindle-An Integration of Wet and Dry Systems Biology

The mitotic spindle is an essential molecular machine involved in cell division, whose composition has been studied extensively by detailed cellular biology, high-throughput proteomics, and RNA interference experiments. However, because of its dynamic organization and complex regulation it is difficult to obtain a complete description of its molecular composition. We have implemented an integrated computational approach to characterize novel human spindle components and have analysed in detail the individual candidates predicted to be spindle proteins, as well as the network of predicted relations connecting known and putative spindle proteins. The subsequent experimental validation of a number of predicted novel proteins confirmed not only their association with the spindle apparatus but also their role in mitosis. We found that 75% of our tested proteins are localizing to the spindle apparatus compared to a success rate of 35% when expert knowledge alone was used. We compare our results to the previously published MitoCheck study and see that our approach does validate some findings by this consortium. Further, we predict so-called “hidden spindle hub”, proteins whose network of interactions is still poorly characterised by experimental means and which are thought to influence the functionality of the mitotic spindle on a large scale. Our analyses suggest that we are still far from knowing the complete repertoire of functionally important components of the human spindle network. Combining integrated bio-computational approaches and single gene experimental follow-ups could be key to exploring the still hidden regions of the human spindle system.

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen‐capturing cell towards a professional antigen presenting cells. In this study, a 2‐D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14+ peripheral blood monocytes was investigated using two pH ranges (pH 4–7 and 6–9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein‐interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune‐associated disorders.